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Cell-Based In Vitro Assay Automation: Balancing Technology and Data Reproducibility/Predictability.

Brande Thomas-Fowlkes1,2, Steven Cifelli1, Sarah Souza3,4

  • 1Screening, Target and Compound Profiling, Merck Research Labs, Merck & Co., Inc., Kenilworth, NJ, USA.

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|February 1, 2020
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Summary
This summary is machine-generated.

The BlueWasher method for removing medium in G-protein-coupled receptor (GPCR) assays improves potency measurements and reduces false negatives. This noncontact centrifugation technique enhances assay performance and reproducibility for drug discovery efforts.

Keywords:
BlueWasherGPCRHTSIP-One assayhigh-throughput screening

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Area of Science:

  • Pharmacology and Drug Discovery
  • Cell-Based Assays
  • Biochemical Assays

Background:

  • G-protein-coupled receptors (GPCRs) are crucial targets for drug development, with many existing drugs modulating their activity.
  • Homogenous time-resolved fluorescence (HTRF) inositol monophosphate (IP-1) assays are standard for profiling GPCR targets at Merck Research Laboratories (MRL).
  • Discrepancies in in vitro assays supporting structure-activity relationship (SAR) studies necessitate evaluation of different assay methodologies.

Purpose of the Study:

  • To evaluate alternative assay paradigms for removing growth medium prior to compound addition in IP-1 accumulation assays.
  • To assess the impact of a noncontact centrifugation method (BlueWasher) compared to traditional manual evacuation.
  • To improve assay reproducibility and data quality for GPCR drug discovery.

Main Methods:

  • Comparison of BlueWasher noncontact centrifugation with manual medium removal in HTRF IP-1 cell-based functional assays.
  • Profiling of GPCR targets using adherent cells in 384-well microplates.
  • Determination of IP-1 accumulation and compound potencies across multiple structural classes.

Main Results:

  • The BlueWasher method resulted in left-shifted potencies for multiple structural classes compared to manual evacuation.
  • The noncontact centrifugation technique successfully rescued "false negatives" observed with the traditional method.
  • Assay performance improved, with the minimum significant ratio for challenging chemotypes decreasing from ~5-6 to <3.

Conclusions:

  • Employing the BlueWasher method offers a significant improvement in GPCR assay performance and data reliability.
  • This technique enhances reproducibility across scientists and sites, particularly for highly protein-bound small molecules.
  • The BlueWasher provides a valuable approach for reducing assay replicates and supporting lead optimization SAR efforts.